NucleoSpin RNA/Protein—Isolation and Purification of RNA & Protein from ONE Lysate

NucleoSpin RNA/Protein is a complete mini kit with recombinant DNase and shredders for rapid, high-yield isolation and purification of total RNA and protein from cells and tissue. This kit allows simultaneous isolation of RNA and protein from a single lysate in one working procedure, from a wide variety of starting materials. The simple, fast, and environmentally friendly column-based procedure does not use phenol, chloroform, or acetone.

This method is very useful for researchers interested in gene regulation mechanisms, such as knockdown of mRNA expression by siRNAs and its resulting influence on protein expression. Such studies are often complicated by small sample sizes and the need to use differing and often incompatible techniques to obtain RNA and protein. Dividing a sample into several parts for independent purification procedures is difficult especially for small samples (e.g., biopsy material). Moreover, the isolation of RNA and protein from different sample aliquots usually necessitates proof of sample equality because the analysis might be mistrusted if the experimental samples used for nucleic acid and protein purification are not absolutely identical. NucleoSpin RNA/Protein resolves these issues by accommodating small sample sizes and providing compatible techniques for RNA and protein isolation.

Samples are lysed by incubation in a buffer containing large amounts of chaotropic salts. This not only destroys the cell structure of the sample, but simultaneously inactivates enzymes such as RNases, phosphatases, and proteases, and thus prevents RNAs and proteins from being degraded. The buffer dissolves even sparingly soluble proteins virtually quantitatively, and at the same time creates appropriate binding conditions for RNA adsorption to the silica membrane of NucleoSpinRNA/Protein Columns. Thus, RNA and protein from the same sample can be separated by simply centrifuging the lysate through the NucleoSpin RNA/Protein spin column—the RNA is bound to the membrane while the protein is resolved in the column flowthrough.

* The amount of DNA contamination is significantly reduced during on-column rDNase digestion. However, in very sensitive applications it might be possible to detect traces of DNA. The NucleoSpin RNA/Protein system is checked by the following procedure: One million HeLa cells are subjected to RNA isolation according to the protocol. RNA eluate is used as template for PCR detection of a 1 kb fragment in a 30 cycle reaction. Generally, no PCR fragment is obtained if the rDNase is applied. However, a strong PCR fragment is obtained if rDNase is omitted. The eventuality of DNA detection with PCR increases with:

The number of DNA copies per preparation: Single copy target < plastidial/ mitochondrial target < cells transfected with plasmid

Decreasing PCR amplicon size

Applications

Rapid isolation and purification of total RNA and protein from cultured cells and tissue